This invention relates generally to rate nephelometric techniques for analyzing precipitate forming reactions between antigens and antibodies and particularly to improved techniques for determining the rate from antigen-antibody precipitates by providing a variable time for verifying peak rates, clearing the system after determining the peak rate for a sample and terminating the antigen excess reaction after the rate exceeds a threshold value.
Certain analytes such as proteins in body fluids may be detected by monitoring chemical reactions between the analytes and antibodies produced in goats, rabbits, etc. In particular, polyvalent protein antigens in sera may react with their corresponding antibodies to produce precipitates. Such antibodies are called precipitins, and their reactions are called immunoprecipitin reactions. In these reactions the amount of precipitate is a function of either the antibody concentration or the antigen concentration, depending upon the relative concentrations of the antigen and antibody.
Nephelometry involves measuring the intensity of light scattered by particles in suspension in a cell when a beam of light is passed through the cell. Rate nephelometry is the monitoring of the rate of change of the amount of light scattered as a reaction proceeds. The resulting complexing and consequent changing scattered light intensity occurs at a rate that increases gradually at first. The rate then increases rapidly until it reaches a peak rate before it decreases to zero as either the antibody or the antigen is depleted.
The analyzer electronics derives the peak value of the rate of change from the scattered light signal. For these purposes, the antigen-antibody immunoprecipitin reaction is conducted in an optically transparent sample container or vial. An excitation system directs a beam of light into the sample container, and a detection system measures light scattered at a forward angle from the precipitate. The detected nephelometric, or light scatter, signal is differentiated to provide a function indicative of the rate. The peak value of the rate is indicative of the concentration of the desired antigen or antibody.
Although less sensitive than enzyme immunoassay, fluorescent immunoassay and radioimmunoassay, nephelometry and rate nephelometry provide the most convenient and direct method for measuring most clinically significant proteins. Nephelometric measurements require no labels and provide direct real time monitoring of the antigen-antibody reaction.
The basis for nephelometric determination of antigens and antibodies is the formation of molecular aggregates when the bivalent antibody molecules combine with multivalent antigen molecules. When the concentrations of the antigens and antibodies are near equivalence, considerable cross linking occurs between the molecules. Antibody molecules bridge between antigen molecules to link several antigen molecules and many antibody molecules into large molecular aggregates that form a precipitate.
These molecular aggregates, after attaining molecular weights of about 3 million or greater, scatter an appreciable amount of light, which may be monitored with various means for detecting light. When the antibody is present in considerable excess, only small scattering centers develop because each antigen tends to have its sites saturated with antibody molecules. The probability that a single antibody molecules will form a bridge between two antigen molecules is small. The reaction forms complexes of the form Ag(Ab).sub.m, where Ag represents the antigen, Ab represents the antibody and m is the valency of the antigen; but larger complexes do not form. No precipitation occurs in extreme antibody excess.
In the case of antigen excess, each antibody molecule has both of its sites occupied by different antigen molecules. Complexes of the form (Ag).sub.2 Ab form, but there are insufficient antibody molecules to bridge between the antigen molecules to form a cross linked lattice.
In the case of antibody excess, or low antigen concentration, no free antigen molecules appear in the supernatant, and an increasing amount of precipitation occurs as antigen is added to the sample. On a plot of peak rate versus antigen concentration, the peak rate increases from zero to a maximum and then decreases from the maximum with further increases in antigen concentration. On the ascending portion of the curve, at lower antigen concentrations, there is an excess of antibody. As further antigen is added, the system moves into antigen excess such that the antigen ties up all the antibody molecules without cross linking. There is a decrease in the total amount of precipitation, and no free antibody is found in the supernatant.
The doubled valued nature of the functional relationship between the antigen concentration and the amount of precipitate formed presents problems in measurement because a given amount of precipitate can correspond to both a low amount and a high amount of antigen. The measuring range in nephelometry is preferably on the ascending region of the curve where there is an excess amount of antibody. Only measurements in the ascending region of the curve provide reliable data on the amount of antigen present in a sample. Clinical applications of nephelometry generally require analysis of a large number of samples. Therefore, the time required for measuring each sample is an important consideration.
After a measurement has been made in the ascending portion of the curve, it is necessary to verify that the peak rate obtained was valid. The time required for this verification is called the peak verify time. A low scatter signal may represent either a low antigen concentration in the sample or an antigen excess condition. Therefore, it is necessary to test each verified peak rate to determine whether it corresponds to antigen excess or antibody excess.
If it is determined that a measured peak rate corresponds to a higher antigen concentration on the descending portion of the curve in antigen excess, it is necessary to dilute the sample and remeasure the rate for the sample. The dilutions and remeasuring are repeated until a peak rate in antibody excess is obtained. After an acceptable peak rate measured in antibody excess is derived from the diluted sample, the corresponding antigen concentration is scaled upward by the appropriate dilution factor to determine the actual antigen concentration of the original sample.
There are several publications describing nephelometric assay of antigen-antibody reactions and addressing the problems encountered in determining the antigen or antibody excess condition of such reactions. These publications include: (1) Savory et al., Kinetics of the IgG-anti-IgG Reaction as Evaluated by Conventional and Stopped-flow Nephelometry, Clin. Chem., 20, 1071 (1974); (2) Buffone et al., Use of a Laser-equipped Centrifugal Analyzer for Kinetic Measurement of Serum IgG. Clin. Chem., 20, 1320 (1974); (3) Buffone et al., Evaluation of Kinetic Light Scattering as an Approach to the Measurement of Specific Proteins With the Centrifugal Analyzer. I. Methodology. Clin. Chem., 21. 1731 (1975); (4) Buffone et al., Evaluation of Kinetic Light Scattering as an Approach to the Measurement of Specific Proteins With the Centrifugal Analyzer. II. Theoretical Considerations. Clin. Chem., 21, 1735 (1975); (5) Tiffany et al., Specific Protein Analysis by Light-scatter Measurement With a Miniature Centrifugal Fast Analyzer, Clin. Chem., 20, 1055 (1974); (6) Anderson et al., A Rate Nephelometer for Immunoprecipitin Measurement of Specific Serum Proteins in Automated Immunoanalysis, 2, R. F. Ritchie, Ed., Marcel Dekker, N.Y. (1978), pp 409-469; and (7 ) Sternburg, Monitoring the Precipitin Reaction Using Rate Nephelometry, ACPR27, April, (1984).
Savory et al., Clin. Chem., 20, 1071 (1974) and Buffone et al., Clin. Chem., 20, 1320 (1974) disclose a two-point semi-kinetic method for measuring specific proteins by deriving the average rate of change of scatter between two fixed times. These references disclose the scatter intensity rises more rapidly in comparison with the end value that it approaches in antigen excess than in antibody excess. These references neither disclose nor suggest any method for utilizing such behavior for determining an excess of antibody or antigen.
Buffone et al., Clin. Chem., 21. 1731 (1975) and Buffone et al., Clin. Chem., 21, 1735 (1975) disclose that consideration of later time intervals with the use of both PBS and PEG-PBS demonstrate no unique characteristics on which differentiation of either antigen or antibody excess samples could be used and that the kinetic procedure cannot directly detect antigen excess. Therefore, although the fundamental properties of antigen-antibody reactions are disclosed, these references fail to disclose kinetic methods for determining antigen or antibody excess.
Tiffany et al., Clin. Chem., 20, 1055 (1974) reports a study of kinetic and equilibrium measurement of antigen-antibody reactions and the achievement of better precision with equilibrium measurements. Tiffany et al. disclose a method for determining antigen excess for equilibrium measurements by measuring a change in equilibrium light scatter intensity caused by the post-addition of a small quantity of antibody into the reaction cell after the primary antigen-antibody reaction has reached equlibrium. If the primary antigen-antibody reaction proceeded in an antigen excess condition, and additional antibody is injected into the reaction cell containing the equilibrated reaction components, the excess antigen reacts with the injected antibody and produces a significant change in scatter intensity. On the other hand, if the primary antigen-antibody reaction proceeded in antibody excess, subsequent injection of the additional antibody produces an insignificant response.
Although determination of antigen or antibody excess by post addition of reactant into the primary reaction is a reliable technique, it is time consuming to perform. Consequently, a time delay is introduced while waiting for the primary reaction to reach equilibrium before the post addition step.
U.S. Pat. No. 4,157,871 to Anderson et al. discloses several kinetic methods for determining antigen excess. In one such method, the peak rate value and the elapsed time from the start of a reaction to occurrence of the peak rate are graphed as functions of increasing antigen concentration for a fixed antibody concentration. A coordinate transformation is used to derive a single valued function, derived from the peak rate and the time thereto, for distinguishing antigen excess.
In a second method disclosed by U.S. Pat. No. 4,157,871 the rate signal, which is the first derivative of the nephelometric signal, is diffentiated to generate the second derivative of the nephelometric signal. The elapsed time from the start of the reaction to the occurrence of the peak of the rate signal is determined together with the time difference between the peak value of the rate signal and the peak value of the second derivative signal. A ratio that distinguishes between antigen excess and antibody excess is established by dividing the elapsed time to the peak rate by the time difference between the peak values of the first and second derivative signals.
U.S. Pat. No. 4,204,837 to Sternberg et al. discloses a method of nephelometric analysis of antigen-antibody reactions to determine whether the reaction is in an antigen excess or antibody excess condition. A first reaction between antigen and antibody reaction components is initiated, and the rate of change of a nephelometric signal is derived from the reaction to develop a rate signal. The peak value of the rate signal provides a measure of the antigen concentration. Sternberg et al. disclose that the rate signal provides kinetic information for may samples from which the antigen or antibody excess condition of the first reaction can be determined without requiring a further step of post-addition of antigen or antibody to the reaction.
Sternberg et al. disclose normal measuring range of peak values between upper and lower thresholds that defines an ambiguous zone for peak values for which a reaction may be in either antigen or antibody excess. Samples having peak heights greater than the threshold can immediately be eliminated for being greater than the normal measuring range, i.e. rejected as being clearly in antigen excess, or as being in near equivalence whether on the antibody or antigen side of the kinetic equivalence point. Sternberg et al. disclose that samples having a peak height lower than the lower threshold are regarded as being clearly on the antibody excess portion of the response curve since it is unlikely that antigen excess samples in a physiological feasible range will exhibit peak heights lower than the lower threshold. The method of Sternberg et al. does not require a post addition step for samples exhibiting peak values below the lower threshold for determining the antigen or antibody excess condition. Similarly, samples exhibiting peak heights above the threshold do not require post-addition. Such samples are rejected, and the reaction is repeated at a higher dilution, or lower concentration, of antigen. The step of post addition for determining the antigen or antibody excess condition is required only for samples having peak heights in the ambiguous zone.
The time required for occurrence of the peak rate depends upon the antigen concentration. For typical concentrations encountered in clinical applications, the peak rate occurs in a time range of about ten to sixty seconds. Rate nephelometry for quantitative measurement of specific proteins uses a constant peak verify time over the entire measuring range of the assay. However, the time actually required to verify that a measured peak rate is indeed the peak of interest, depends upon the rate. Higher rates require less time for verification than lower rates because higher rates have higher signal to noise ratios than lower rates. Therefore, prior peak verify techniques require excessive amounts of time and reduce the throughput of nephelometric measurement systems.